Complex Molecular Evolutionary Models and Information Theoretic Approaches Provide Genomic Perspectives on Amphibian Evolution

Paul M. Hime 16 May, 2017 Blue Waters Symposium [email protected] The Evolution of Life on Earth

• All life traces its origins back to a single common ancestor nearly 4 billion years ago

• But today, there are tens of millions of species!

• Reconstructing the genealogy of life is fundamental to nearly all areas of modern biology. The Evolution of Life on Earth

“Nothing in biology makes sense, except in light of evolution” Dobzhansky

“Nothing in evolutionary biology makes sense, except in light of phylogeny” All Organisms on Earth Trace Their Origins Back to a Single Common Ancestor Genomes Are Documents of Evolutionary History Organisms’ Genomes Evolve through Time Phylogenetic Reconstruction

• Phylogenies are hypotheses about ancestor - descendent relationships.

• These can be estimated from genetic data (in the context of a model).

• Simple case: enumerate all possible trees, pick the “best”.

• Tree space explodes factorially with increasing numbers of taxa.

• Use heuristic search strategies to explore tree- and parameter-space. Models in Evolutionary Biology

• Evolutionary biology is an inherently historical discipline.

• In evolutionary biology, one cannot “replay the tape” of life...

• We use statistical approaches to compare competing sets of models, in the light of data which we collect.

“All models are wrong. Some are useful.”

George Box Data ≠ Information (Except in the Context of an Appropriate Model)

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Species 1 ACCGAGGGCATCGATCGACTACCTTAGGGCTCTAGCCTGTTTCGTGCTAGCTGACTGATCGTAGTGTAGCTGACTGTGTG Species 2 ACCGAGGGCTTCGATCGACTACCTTAGGGCTCTAGCCTGTTACGTGCTAGCTGACTGATCGTAGTGTAGCTGACTGTGTG Species 3 ACCGAGGGCATCGATCGACTACCTTAGGGCTCTAGCCTGTTACGTGCTAGCTGACTGATCGTAGTGTAGCTGACTGTGTG Species 4 ACCGAGGGCTTCGATCGACTACCTTAGGGCTCTAGCCTGTTACGTGCTAGCTGACTGATCGTAGTGTAGCTGACTGTGTG Species 5 ACCGAGGGCTTCGATCGACTACCTTAGGGCTCTAGCCTGTTACGTGCTAGCTGACTGATCGTAGTGTAGCTGACTGTGTG Species 6 ACCGAGGGCATCGATCGACTACCTTAGGGCTCTAGCCTGTTTCGTGCTAGCTGACTGATCGTAGTGTAGCTGACTGTGTG Species 7 ACCGAGGGCTTCGATCGACTACCTTAGGGCTCTAGCCTGTTCCGTGCTAGCTGACTGATCGTAGTGTAGCTGACTGTGTG Species 8 ACCGAGGGCTTCGATCGACTACCTTAGGGCTCTAGCCTGTTTCGTGCTAGCTGACTGATCGTAGTGTAGCTGACTGTGTG Species 9 ACCGAGGGCATCGATCGACTACCTTAGGGCTCTAGCCTGTTTCGTGCTAGCTGACTGATCGTAGTGTAGCTGACTGTGTG Species 10 ACCGAGGGCTTCGATCGACTACCTTAGGGCTCTAGCCTGTTTCGTGCTAGCTGACTGATCGTAGTGTAGCTGACTGTGTG Species 11 ACCGAGGGCTTCGATCGACTACCTTAGGGCTCTAGCCTGTTTCGTGCTAGCTGACTGATCGTAGTGTAGCTGACTGTGTG Species 12 ACCGAGGGCTTCGATCGACTACCTTAGGGCTCTAGCCTGTTCCGTGCTAGCTGACTGATCGTAGTGTAGCTGACTGTGTG Species 13 ACCGAGGGCATCGATCGACTACCTTAGGGCTCTAGCCTGTTTCGTGCTAGCTGACTGATCGTAGTGTAGCTGACTGTGTG Species 14 ACCGAGGGCTTCGATCGACTACCTTAGGGCTCTAGCCTGTTTCGTGCTAGCTGACTGATCGTAGTGTAGCTGACTGTGTG Species 15 ACCGAGGGCTTCGATCGACTACCTTAGGGCTCTAGCCTGTTTCGTGCTAGCTGACTGATCGTAGTGTAGCTGACTGTGTG Species 16 ACCGAGGGCCTCGATCGACTACCTTAGGGCTCTAGCCTGTTTCGTGCTAGCTGACTGATCGTAGTGTAGCTGACTGTGTG Species 17 ACCGAGGGCTTCGATCGACTACCTTAGGGCTCTAGCCTGTTCCGTGCTAGCTGACTGATCGTAGTGTAGCTGACTGTGTG Species 18 ACCGAGGGCCTCGATCGACTACCTTAGGGCTCTAGCCTGTTTCGTGCTAGCTGACTGATCGTAGTGTAGCTGACTGTGTG Species 19 ACCGAGGGCTTCGATCGACTACCTTAGGGCTCTAGCCTGTTTCGTGCTAGCTGACTGATCGTAGTGTAGCTGACTGTGTG Species 20 ACCGAGGGCTTCGATCGACTACCTTAGGGCTCTAGCCTGTTGCGTGCTAGCTGACTGATCGTAGTGTAGCTGACTGTGTG Species 21 ACCGAGGGCCTCGATCGACTACCTTAGGGCTCTAGCCTGTTTCGTGCTAGCTGACTGATCGTAGTGTAGCTGACTGTGTG Species 22 ACCGAGGGCTTCGATCGACTACCTTAGGGCTCTAGCCTGTTGCGTGCTAGCTGACTGATCGTAGTGTAGCTGACTGTGTG Species 23 ACCGAGGGCTTCGATCGACTACCTTAGGGCTCTAGCCTGTTTCGTGCTAGCTGACTGATCGTAGTGTAGCTGACTGTGTG Species 24 ACCGAGGGCTTCGATCGACTACCTTAGGGCTCTAGCCTGTTTCGTGCTAGCTGACTGATCGTAGTGTAGCTGACTGTGTG * * informative site informative site Models of Nucleotide Substitution

Gamma-distributed rate heterogeneity across sites (discretized in practice) Codon-Based Models of Molecular Evolution The Multispecies Coalescent Model

Gene divergences always predate species divergences

Stochastic coalescent processes can lead to gene tree / species tree discordance

Modified from Leliaert et al. 2014 Gene Tree - Species Tree Discordance Population-Level Processes Affect The Expected Distributions of Gene Coalescence

Luay Nakhleh

Conflicting phylogenetic signal from different loci is expected, especially for more recent divergence events and large effective population sizes.

What about at deep scales?

Many loci (regions of the genome) may be needed for difficult questions. The Genomics Revolution in Evolutionary Biology

It has never been easier to collect genomic data in non-model organisms. The Genomics Revolution in Evolutionary Biology

It has never been easier to collect It has never been genomic data in easier to collect non-model genomic data in organisms. non-model organisms. Data ≠ Information (Except in the Context of an Appropriate Model) Data ≠ Information (Except in the Context of an Appropriate Model) Data ≠ Information (Except in the Context of an Appropriate Model) Data ≠ Information (Except in the Context of an Appropriate Model) Some of the Many Tradeoffs in Phylogenomics

The genomic revolution is now offering unprecedented opportunities to tackle thorny questions in evolutionary biology.

But these opportunities bring analytical and computational costs. Some of the Many Tradeoffs in Phylogenomics

The genomic revolution is now offering unprecedented opportunities to tackle thorny questions in evolutionary biology.

But these opportunities bring analytical and computational costs. Genomic perspectives on the amphibian tree of life • Amphibians provide a rich system for testing phylogenetic hypotheses at both deep and shallow scales of divergence.

• What is the topology of the amphibian Tree of Life???

• How confident are we in that topology?

• What are the inter-ordinal relationships?

• What is the nature of support across the genome?

• What are “best” practices with large phylogenomic data sets??? Extant Amphibian Diversity

Amphibians - 7,660 species as of 28 April, 2017 (http://AmphibiaWeb.org) The three amphibian orders likely diverged 300+ MYA.

Map from BiodiversityMapping.org Extant Amphibian Diversity Caecilians (Gymnophiona) - 205 species, 33 genera, 10 families

Map from BiodiversityMapping.org Extant Amphibian Diversity Salamanders (Caudata) - 695 species, 68 genera, 10 families

Map from BiodiversityMapping.org Extant Amphibian Diversity Frogs and Toads (Anura) - 6,760 species, 448 genera, 55-65 families

Map from BiodiversityMapping.org Amphibian Relationships Hundreds of studies have addressed phylogenetic affinities of amphibians... Amphibian Relationships Hundreds of studies have addressed phylogenetic affinities of amphibians...

This project aims to sample hundreds of nuclear genes for hundreds of amphibian species Inter-Ordinal Amphibian Relationships

Three main hypotheses for phylogenetic relationships among the three amphibian orders (assuming that Amphibia is monophyletic...)

Batrachia Acauda Procera

The potential resolutions of these deep branches each have very different implications for our understanding of amphibian evolution. Inter-Ordinal Amphibian Relationships

• There are 15 possible topologies for the three amphibian orders and amniotes,Inter-Ordinal assumingAmphibian Relationships that Amphibia may or may NOT be monophyletic.. Three main hypotheses for phylogenetic relationships among the three amphibian orders (assuming that Amphibia is monophyletic...)

Batrachia Acauda Procera

These three orders likely diverged ~300 million years ago. Taxonomic Sampling across Amphibia

• We targeted 325 amphibian taxa (296 “worked”).

• 276 genera (> 50% of recognized genera)...

• 96% of recognized families (and most subfamilies)...

• Taxa were sampled roughly in proportion to species richness. Taxonomic Sampling across Amphibia

• We targeted 325 amphibian taxa (296 “worked”).

• 276 genera (> 50% of recognized genera)...

• 96% of recognized families (and most subfamilies)...

• Taxa were sampled roughly in proportion to species richness.

• Multiple outgroups were included to root the phylogeny.

Anolis Chrysemys Gallus Homo Latimeria Generating Genomic Data in Amphibians

• We developed an amphibian-specific gene capture system which targets 366 semi-conserved nuclear exons.

• Probes were designed from genomic/transcriptomic* resources for:

1 Caecilian:

Ichthyophis

4 Salamanders:

Ambystoma* Desmognathus Cryptobranchus* Notophthalmus* 6 Frogs:

Gastrophryne Pseudacris Xenopus Rana (Silurana) Ascaphus Mixophyes (Lithobates) Nuclear Gene Tree Estimation

• Independently estimated ML gene trees for all genes in RAxML.

• Used separate (best-fit) partitioning schemes and nucleotide substitution models for each gene (GTR+G).

• Conducted 500 non-parametric bootstrap replicates (across sites) to assess “support” for branches in these gene trees.

• Identified outlier taxa for each gene (and removed those taxa).

• Conducted analyses with unconstrained and constrained topological backbones. Species Tree Estimation

• Use multiple algorithms to estimate the topology of the species tree.

• “Shortcut” methods attempt to reconcile collections of gene trees into an estimate of the species tree (Astral and MulRF).

+ + + + ... ®

• Also implemented a gene-tree-free approach (SVDQuartets).

• Lastly, estimated a concatenated ML tree in RAxML, using a best-fit partitioning scheme of 76 distinct partitions (separate GTR+G models).

• Used the non-parametric bootstrap across sites to assess “support” (sites and genes for Astral). Brief Overview of Results • Nearly all families are recovered as monophyletic.

• Shallow-scale relationships are largely in line with previous studies.

• Most higher-order clades are recovered.

• Deep branches receive strong bootstrap support in species tree analyses... Astral2"TUSBM4QFDJFT5SFF&TUJNBUFEGSPN"ODIPSFE-PDJ "NQIJCJBBOE&BDI0SEFS$POTUSBJOFEUP#F.POPQIZMFUJD Species Tree

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I4408_MVZ_226277_Anura_Megophryidae_Brachytarsophrys_feae_seq1

I4409_CAS_240922_Anura_Megophryidae_Megophrys_glandulosa_seq1 I6416_AMCC_144796_Anura_Megophryidae_Ophryophryne_hansi_seq1

I6473_CAS_234295_Anura_Megophryidae_Scutiger_gongshanensis_seq1

I9326_CR04_Labisko_Anura_Sooglossidae_Sooglossus_sechellensis_seq1 I6425_AMCC_106489_Anura_Megophryidae_Leptolalax_bourreti_seq1

I4384_PMH_01_Anura_Calyptocephalellidae_Calyptocephalella_gayi_seq1

I4399_SANBI_1954_Anura_Heleophrynidae_Heleophryne_purcelli_seq1

I13524_REF_MixSch_Anura_Myobatrachidae_Mixophyes_schevilli_seq1

I6486_SAMAR66870_Anura_Myobatrachidae_Lymnodynastes_dumerilli_seq1

I8564_MV_21476_Anura_Myobatrachidae_Geocrinia_victoriana_seq1I8571_MV_21479_Anura_Myobatrachidae_Paracrinia_haswelli_seq1

I8570_MV_21528_Anura_Myobatrachidae_Neobatrachus_sudelli_seq1

I4422_MVZ_164829_Anura_Rhinodermatidae_Rhinoderma_darwinii_seq1

I8561_MV_18153_Anura_Myobatrachidae_Crinia_signifera_seq1

I4371_Cab_381_Anura_Cycloramphidae_Cycloramphus_cavagua_seq1

I9034_70661_Anura_Myobatrachidae_Notaden_nichollsi_seq1

I4377_MVZ_164828_Anura_Batrachylidae_Batrachyla_taeniata_seq1

I4427_KU_290640_Anura_Telamatobiidae_Telmatobius_niger_seq1 I4372_MVZ_188060_Anura_Alsodidae_Alsodes_gargola_seq1 I4411_KZ1713_Anura_Hylodidae_Hylodes_phyllodes_seq1 I4388_MVZ_247561_Anura_Ceratophryidae_Ceratophrys_cornuta_seq1 I4441_YPM_013120_Anura_Ceratophryidae_Lepidobatrachus_laevis_seq1

I4434_AMCC_125581_Anura_Ceratophryidae_Chacophrys_pierottii_seq1I4400_BPN_1286_Anura_Hemiphractidae_Stefania_evansi_seq1

I4158_QCAZA48818_Anura_Hylidae_Phyllomedusa_vaillantii_seq1

• High bootstrap I4157_QCAZA48552_Anura_Hylidae_Cruziohyla_calcarifer_seq1

I8568_PMH_Litoria2014_Anura_Hylidae_Litoria_caerulea_seq1 I8569_LSUMNS_9884_Anura_Hylidae_Litoria_thesaurensis_seq1

I4439_YPM_010666_Anura_Hylidae_Hypsiboas_crepitans_seq1

I6442_CAS_245062_Anura_Hylidae_Sphaenorhynchus_lacteus_seq1I4160_QCAZA51852_Anura_Hylidae_Hyloscirtus_palmeri_seq1

I4413_MVZ_234650_Anura_Pelobatidae_Pelobates_syriacus_seq1 I4414_MVZ_186009_Anura_Pelodytidae_Pelodytes_ibericus_seq1 I4424_CAS_229217_Anura_Scaphiopodidae_Scaphiopus_couchii_seq1 I6469_MVZ_145187_Anura_Scaphiopodidae_Spea_hammondii_seq1

I6453_PMH_2014_Anura_Pipidae_Hymenochirus_boettgeri_seq1

Xenopus_TAXID_8364_seq1

I6444_MVZ_247511_Anura_Pipidae_Pipa_pipa_seq1 I6471_MVZ_247548_Anura_Hylidae_Trachycephalus_coriaceus_seq1 I4423_MVZ_164756_Anura_Rhinophrynidae_Rhinophrynus_dorsalis_seq1 I4397_MVZ_235689_Anura_Discoglossidae_Discoglossus_pictus_seq1

I4373_MVZ_231914_Anura_Alytidae_Alytes_obstetricans_seq1

I8555_CAS_242112_Anura_Bombinatoridae_Bombina_microdeladigitora_seq1 I6468_MVZ_257781_Anura_Hylidae_Scinax_staufferi_seq1 I4448_DMG_5134_Anura_Leiopelmatidae_Leiopelma_hochstetteri_seq1 support across I13520_REF_AscMon_Anura_Ascaphidae_Ascaphus_montanus_seq1 I3715_JK03_Caudata_Plethodontidae_Desmognathus_fuscus_seq1 I6482_MCZ_A_148702_Anura_Hylidae_Osteopilus_dominicensis_seq1I4169_QCAZA53552_Anura_Hylidae_Nyctimantis_rugiceps_seq1 I3716_JK08_Caudata_Plethodontidae_Desmognathus_quadromaculatus_seq1

I3711_JK07_Caudata_Plethodontidae_Desmognathus_wrighti_seq1

100

100 I4359_P_82_Caudata_Plethodontidae_Phaeognathus_hubrichti_seq1 I8560_CHUNB64717_Anura_Hylidae_Corythomantis_greeningei_seq1 I3710_CSU01_Caudata_Plethodontidae_Aneides_flavipunctatus_seq1 I6462_MVZ_264263_Anura_Hylidae_Dendropsophus_microcephalus_seq1 84 I3717_JK09_Caudata_Plethodontidae_Plethodon_jordani_seq1

I12498_MVZ_247157_Caudata_Plethodontidae_Karsenia_koreana_seq1 100 100

100 I4358_AMCC_118113_Caudata_Plethodontidae_Bolitoglossa_riletti_seq1 100

100 I12496_MVZ_263972_Caudata_Plethodontidae_Nyctanolis_pernix_seq1

100

100 I11148_ELJ_1554_Caudata_Plethodontidae_Batrachoseps_nigriventris_seq1

100 I8572_LSUMNS_12511_Anura_Pseudidae_Pseudis_paradoxa_seq1 I12499_Tpeirson_03_Caudata_Plethodontidae_Hemidactylium_scutatum_seq1

100 100 I9336_PMH_15_APR_2013_Caudata_Plethodontidae_Gyrinophilus_porphyriticus_seq1 100 I12497_PMH_PR02_Caudata_Plethodontidae_Pseudotriton_ruber_seq1 100 100 the tree I6427_AMCC_125603_Anura_Hylidae_Triprion_petasatus_seq1 I9327_JRJ_2012_Caudata_Plethodontidae_Eurycea_lucifuga_seq1 69.6667 100 42 100 I4351_MVZ_232868a_Caudata_Amphiumidae_Amphiuma_tridactylum_seq1

I3536_LSUMZ_H_11333_Caudata_Rhyacotritonidae_Rhyacotriton_olympicus_seq1 100 I4447_YPM_014191_Anura_Hylidae_Smilisca_fodiens_seq1 100 100 I6431_AMCC_117944_Anura_Hylidae_Plectrohyla_matudai_seq1 I4362_MVZ_244076_Caudata_Proteidae_Proteus_anguinus_seq1 100 100 I3535_PMH_7759_Caudata_Proteidae_Necturus_maculosus_seq1 100 99.3333 I9340_TP26609_Caudata_Salamandridae_Triturus_vulgaris_seq1

51 I13522_REF_PseNig_Anura_Hylidae_Pseudacris_nigrita_seq1 100 I9338_TP27066_Caudata_Salamandridae_Neurergus_crocatus_seq1 100 I4446_YPM_013066_Anura_Dendrobatidae_Phyllobates_vittatus_seq1I13521_REF_PseFer_Anura_Hylidae_Pseudacris_feriarum_seq1 I9339_TP24839_Caudata_Salamandridae_Paramesotriton_hongkongensis_seq1 100 I9330_TP24749_Caudata_Salamandridae_Cynops_ensicauda_seq1 I6449_ITF_2014_Anura_Dendrobatidae_Dendrobates_leucomelas_seq1 100 I3534_LSUMZ_H_11856_Caudata_Salamandridae_Notophthalmus_viridescens_seq1 39.3333 I4393_CAS_231821_Anura_Dendrobatidae_Mannophryne_trinitatus_seq1 I9337_TP26195_Caudata_Salamandridae_Echinotriton_chinhaiensis_seq1 I8563_LSUMNS_13667_Anura_Dendrobatidae_Epipedobates_femoralis_seq1 57.6667 100 100 I9333_TP25555_Caudata_Salamandridae_Tylototriton_kweichowensis_seq1 I8559_LSUMNS_16955_Anura_Dendrobatidae_Colostethus_caeruleodactylus_seq1 100 100 100 I9331_TP25088_Caudata_Salamandridae_Salamandra_salamandra_seq1 100 100 I9332_s7539_Caudata_Salamandridae_Salamandrina_terdigitata_seq1 23 61 100 I3544_JK02_Tig_Caudata_Ambystomatidae_Ambystoma_tigrinum_seq1 I12045_QCAZA56305_Anura_Aromobatidae_Allobates_insperatus_seq1 100 100 100 100 I4349_DWW_1781_Caudata_Ambystomatidae_Ambystoma_mexicanum_seq1 100 100 I3541_RB01_OP4_Caudata_Ambystomatidae_Ambystoma_opacum_seq1 I12044_QCAZA44783_Anura_Aromobatidae_Allobates_insperatus_seq1 98 I4195_SBH268267_Anura_Ceuthomantidae_Ceuthomantis_smaragdinus_seq1 100 100 I3538_RB09_T23_Caudata_Ambystomatidae_Ambystoma_talpoidium_seq1 100 I8578_LSUMNS_21241_Anura_Eleutherodactylidae_Syrrhophus_cystignathoides_seq1 100 91.3333 I4356_DWW_2567_Caudata_Dicamptodontidae_Dicamptodon_copei_seq1

100 I13533_REF_SirInt_Caudata_Sirenidae_Siren_intermedia_seq1 100

100 100 100 100 100 100 100 80 99 I3709_PMH_BC16_Caudata_Cryptobranchidae_Cryptobranchus_alleganiensis_seq1 I4398_EMO_01_Anura_Eleutherodactylidae_Eleutherodactylus_coqui_seq1 100 100 22 100 100 100 100 I3707_PMH_ELK13_Caudata_Cryptobranchidae_Cryptobranchus_alleganiensis_seq1 I8556_CFBHT_55_Anura_Brachycephalidae_Brachycephalus_ephippium_seq1 I3704_PMH_C37AF_Caudata_Cryptobranchidae_Cryptobranchus_alleganiensis_seq1

100 98.6667 100 I3703_PMH_AJ12_Caudata_Cryptobranchidae_Andrias_japonicus_seq1 • Inter-ordinal 100 I4391_USMN_534194_Anura_Craugastoridae_Craugastor_noblei_seq1 100 I3702_PMH_AD03_Caudata_Cryptobranchidae_Andrias_davidianus_seq1

I3701_DWW392_Caudata_Hynobiidae_Salamandrella_keyserlingii_seq1 100 I4445_AMCC_107352_Anura_Strabomantidae_Phrynopus_sp_seq1 100 100 100 100 I3542_YPM_9865_Caudata_Hynobiidae_Hynobius_nigrescens_seq1 100 99 100 100 I4426_USNM_268942_Anura_Strabomantidae_Pristimantis_ridens_seq1 100 100 100 92.6667 I3539_YPM_10577_Caudata_Hynobiidae_Pachyhynobius_shangchengensis_seq1 I4380_USMN_533994_Anura_Brachycephalidae_Ischnocnema_ramagii_seq1 I3700_DWW_0379_Caudata_Hynobiidae_Batrachuperus_persicus_seq1 100 68.3333 I4412_MVZ_145208_Anura_Odontophrynidae_Odontophrynus_occidentalis_seq1 93.3333 support is for I6479_SLZ_971026_Gymnophiona_Dermophiidae_Dermophis_mexicanus_seq1 I4386_AMCC_118359_Anura_Centrolenidae_Hyalinobatrachium_fleischmanni_seq1 100 100 I4338_MVZ_228795_Gymnophiona_Dermophiidae_Gymnopis_multiplicata_seq1 100 100 I4339_CAS_218738_Gymnophiona_Dermophiidae_Schistometopum_thomense_seq1

I8558_LSUMNS_17409_Anura_Centrolenidae_Cochranella_adenocheira_seq1 100 I4436_YPM_013118_Gymnophiona_Dermophiidae_Geotrypetes_seraphini_seq1 100 86.6667 100 100 I4346_BPN_Ga169_Gymnophiona_Siphonopidae_Microcaecilia_sp_seq1 I8557_LSUMNS_16979_Anura_Centrolenidae_Centrolene_prosoblepon_seq1 100 100 100 100 99 100 I4347_MVZ_162592_Gymnophiona_Siphonopidae_Siphonops_annulatus_seq1 100 I8567_LSUMNS_15432_Anura_Leptodactylidae_Lithodytes_lineatus_seq1 100 100 100 I4342_MVZ_258024_Gymnophiona_Indotyphlidae_Grandisonia_alternans_seq1 Batrachia 100 I4405_CAS_245125_Anura_Leptodactylidae_Leptodactylus_fuscus_seq1 I4337_BPN_1499_Gymnophiona_Caeciliidae_Caecilia_tentaculata_seq1 100 I4348_MVZ_179733_Gymnophiona_Typhlonectidae_Typhlonectes_natans_seq1 100 100 I4406_MVZ_231766_Anura_Leptodactylidae_Pleurodema_bibroni_seq1 100 100 70.6667 100 I4340_MVZ_179505_Gymnophiona_Herpelidae_Boulengerula_taitana_seq1 18.6667 100 I6441_MVZ_264270_Anura_Leptodactylidae_Physalaemus_pustulosus_seq1 I4437_YPM_013116_Gymnophiona_Herpelidae_Herpele_squalostoma_seq1 100 100 55 100 100 I6458_CAS_231794_Anura_Leptodactylidae_Physalaemus_pustulosus_seq1 I8577_CAS_168812_Gymnophiona_Scolecomorphidae_Scolecomorphus_vittatum_seq1 100 I4345_AMCC_117706_Gymnophiona_Scolecomorphidae_Crotaphatrema_tchabalmbaboensis_seq1 I12500_PMH_2014_redo_Anura_Bufonidae_Melanophryniscus_stelzneri_seq1 100 100 100 100 100 I13518_REF_IchBan_Gymnophiona_Ichthyophiidae_Ichthyophis_bannanicus_seq1 98 I4430_YPM_013728_Anura_Bufonidae_Atelopus_hoogmoedi_seq1 I4343_MVZ_265495_Gymnophiona_Rhinatrematidae_Epicrionops_petersi_seq1 (BS = 100) 100 Gallus_TAXID_9031_seq1 I4383_ECM_4908_Anura_Bufonidae_Anaxyrus_terrestris_seq1 100 100 Chrysemys_TAXID_8478_seq1 100 100 100 85.6667 I8576_LSUMNS_15190_Anura_Bufonidae_Rhinella_marinus_seq1 98.3333 Anolis_TAXID_28377_seq1 100 100 I6467_MVZ_231697_Anura_Bufonidae_Rhamphophryne_macrorhina_seq1 100 Homo_TAXID_9606_seq1 100 Latimeria_TAXID_7897_seq1 I4433_AMCC_105533_Anura_Bufonidae_Capensibufo_rosei_seq1 100 100 I7967_R241_Anura_Rhacophoridae_Rhacophorus_pardalis_seq1 I7581_R690_Anura_Bufonidae_Poyntonophrynus_damaranus_dombensis_seq1 100 100 I4421_CAS_224676_Anura_Rhacophoridae_Rhacophorus_rhodopus_seq1 42 I6481_MCZ_A_139634_Anura_Bufonidae_Nectophryne_batesii_seq1 98 48.3333 I7927_R233_Anura_Rhacophoridae_Feihyla_palpebralis_seq1 95.3333 I6459_CAS_241141_Anura_Rhacophoridae_Polypedates_leucomystax_seq1 I4429_YPM_013738_Anura_Bufonidae_Ansonia_longidigita_seq1 98.3333 I7925_R538_Anura_Rhacophoridae_Chiromantis_xerampelina_seq1 I6464_MVZ_239399_Anura_Bufonidae_Leptophryne_borbonica_seq1 100 100 76.3333 I6456_CAS_233160_Anura_Rhacophoridae_Philautus_parvulus_seq1 100 100 100 69.6667 I7961_R1120_Anura_Rhacophoridae_Raorchestes_gryllus_seq1 I10935_NCBS_AI442_Anura_Nasikabatrachidae_Nasikabatrachus_sahyadrensis_seq1 100 100 100 I7935_R1112_Anura_Rhacophoridae_Kurixalus_appendiculatus_seq1 I13356_PT287_Anura_Microhylidae_Phrynomantis_bifasciatus_seq1

100 100 I7929_R1149_Anura_Rhacophoridae_Gorhixalus_hosii_seq1 I7739_R1330_Anura_Microhylidae_Phrynomantis_annectens_seq1 100 100 I7946_R075_Anura_Rhacophoridae_Nyctixalus_pictus_seq1

I10391_ROM_44169_Anura_Microhylidae_Adelastes_hylonomos_seq1 100 I6470_MVZ_225131_Anura_Rhacophoridae_Theloderma_corticale_seq1

I13354_PT459_Anura_Microhylidae_Otophryne_robusta_seq1 I6440_MVZ_241442_Anura_Rhacophoridae_Buergeria_oxycephalus_seq1 • We’re done, 100 85.3333 100 100 100 I8233_R942_Anura_Mantellidae_Gephyromantis_ambohitra_seq1 I13360_PT271_Anura_Microhylidae_Synapturanus_salseri2_seq1 100 100 I7734_R969_Anura_Mantellidae_Mantidactylus_lugabris_seq1 100 100 100 100 I8562_LSUMNS_17434_Anura_Microhylidae_Ctenophryne_geayi_seq1 I8244_R971_Anura_Mantellidae_Guibemantis_pulcher_seq1 100 I13361_PT198_Anura_Microhylidae_Syncope_carvalhoi_seq1 I7730_R930_Anura_Mantellidae_Mantella_betsileo_seq1 45 I13353_PT340_Anura_Microhylidae_Myersiella_sp_seq1 100 I8229_R928_Anura_Mantellidae_Boophis_albipunctatus_seq1 100 I4407_MVZ_238732_Anura_Mantellidae_Boophis_pyrrhus_seq1 right??!! 43.6667 I13344_PT332_Anura_Microhylidae_Dasypops_schirchi_seq1 98 100 100 100 100 100 I6422_MVZ_238723_Anura_Mantellidae_Aglyptodactylus_madagascariensis_seq1 97.6667 45.3333 100 I7868_R1144_Anura_Ranidae_Hylarana_Sylvirana_nigrovittata_seq1 I13358_PT273_Anura_Microhylidae_Stereocyclops_incrassatus_seq1 100 100 100 100 I13349_PT284_Anura_Microhylidae_Hyophryne_histrio_seq1 100 I6460_CAS_234711_Anura_Ranidae_Pterorana_khare_seq1 100 100 I8575_LSUMNS_10459_Anura_Ranidae_Papurana_papua_seq1 I13337_PT281_Anura_Microhylidae_Arcovomer_sp_seq1 100 100 I6480_YPM_013741_Anura_Ranidae_Hylarana_picturata_seq1 47 100 100 I7908_R1168_Anura_Ranidae_Sanguirana_sanguinea_seq1 100 100 I13347_PT043_Anura_Microhylidae_Hamptophryne_boliviana_seq1 100 71.6667 I13335_PT321_Anura_Microhylidae_Altigius_alios_seq1 100 100 I7849_R185_Anura_Ranidae_Babina_chapaensis_seq1

100 I13526_REF_LitSph_Anura_Ranidae_Lithobates_sphenocephala_seq1 100 I7875_R1141_Anura_Ranidae_Lithobates_pipiens_seq1 100 I4435_AMCC_125588_Anura_Microhylidae_Dermatonotus_muelleri_seq1 85.3333 99.6667 I8574_LSUMNS_17589_Anura_Ranidae_Lithobates_palmipes_seq1 100 I13345_PT059_Anura_Microhylidae_Elachistocleis_helianneae_seq1 I6420_AMCC_138323_Anura_Ranidae_Huia_nasica_seq1

I6466_MVZ_258265_Anura_Ranidae_Odorrana_banaorum_seq1 100 100 I13523_REF_GasCar_Anura_Microhylidae_Gastrophryne_carolinensis_seq1 I6446_CAS_242607_Anura_Ranidae_Amolops_medogensis_seq1 I13357_PT312_Anura_Microhylidae_Scaphiophryne_brevis_seq1 93.3333 79 100 100 I7897_R153_Anura_Ranidae_Pelophylax_ridibunda_seq1 100 100 100 78 I13359_PT265_Anura_Microhylidae_Stumpffia_roseifemoralis_seq1 15 I7882_R1162_Anura_Ranidae_Meristogenys_orphnocnemis_seq1 100

100 I7910_R1164_Anura_Ranidae_Staurois_natator_seq1 I6430_AMCC_103414_Anura_Microhylidae_Stumpffia_grandis_seq1 100 100 I8219_R180_Anura_Dicroglossidae_Nanorana_bourreti_cf_yunnanensis_seq1 100 100 I13336_PT359_Anura_Microhylidae_Anodonthyla_nigrigularis_seq1 I6465_MVZ_231208_Anura_Dicroglossidae_Nanorana_pleskei_seq1 100 99 I6424_AMCC_106520_Anura_Dicroglossidae_Chaparana_Annandia_delacouri_seq1

92.6667 100 • Scrutinize 100 I6417_AMCC_144942_Anura_Dicroglossidae_Quasipaa_verrucospinosa_seq1 100 I4394_CAS_221360_Anura_Dicroglossidae_Limnonectes_kuhlii_seq1 I6435_AMCC_128714_Anura_Microhylidae_Plethodontohyla_notosticta_seq1 100 100

I6436_AMCC_103335_Anura_Microhylidae_Platypelis_occultans_seq1 100 100 99.3333 I7668_R057_Anura_Dicroglossidae_Limnonectes_limborgii_seq1

93.3333 100 I8573_LSUMNS_255_Anura_Ranidae_Limnonectes_limnocharis_seq1

100 100 100 98.6667 I8566_CAS_247917_Anura_Microhylidae_Kalophrynus_pleurostigma_seq1 100 I6419_AMCC_144930_Anura_Dicroglossidae_Fejervarya_limnocharis_seq1

I13350_PT168_Anura_Microhylidae_Kalophrynus_interlineatus1_seq1 100 I7915_R107_Anura_Ranixalidae_Indirana_leithi_seq1 100 100 82 80 100 I6463_MVZ_238744_Anura_Microhylidae_Dyscophus_guineti_seq1 I6450_CAS_243255_Anura_Dicroglossidae_Euphlyctis_cyanophlyctis_seq1 100

100 100 I6452_CAS_241469_Anura_Dicroglossidae_Hoplobatrachus_rugulosus_seq1 100 I6455_CAS_247906_Anura_Microhylidae_Micryletta_inornata_seq1 90.3333 100 I4410_CAS_220433_Anura_Micrixalidae_Micrixalus_borealis_seq1

66.3333 phylogenetic 100 100 I7740_R1208_Anura_Microhylidae_Ramanella_variegata_cf_seq1 100 I4395_CAS_239527_Anura_Dicroglossidae_Occidozyga_lima_seq1 100 100 100 I6472_CPM_2014_Anura_Ceratobatrachidae_Ceratobatrachus_guentheri_seq1 93.3333

85 100 I6418_AMCC_125415_Anura_Ceratobatrachidae_Discodeles_bufoniformis_seq1 I13352_PT236_Anura_Microhylidae_Metaphrynella_sundana_seq1 100 100 I4431_AMCC_125449_Anura_Ceratobatrachidae_Batrachylodes_vertebralis_seq1 100 I4440_YPM_013065_Anura_Microhylidae_Kaloula_pulchra_seq1 70 I4387_CAS_237845_Anura_Ceratobatrachidae_Platymantis_pelewensis_seq1 100 I7654_R059_Anura_Dicroglossidae_Ingerana_sp_nov_2_seq1

100 I7917_R1145_Anura_Ranixalidae_Micrixalus_sp_seq1 100 100 I10392_FMNH_231112_Anura_Microhylidae_Chaperina_fusca_seq1 100 100 I10934_NCBS_AG004_Anura_Nyctibatrachidae_Nyctibatrachus_petraeus_seq1 100 100 I6454_CAS_233947_Anura_Microhylidae_Microhyla_ornata_seq1 I6433_AMCC_105565_Anura_Pyxicephalidae_Strongylopus_bonaespei_seq1

100 98.6667 100 100 I7822_R831_Anura_Pyxicephalidae_Strongylopus_fasciatus_seq1

I13339_PT164_Anura_Microhylidae_Calluella_yunnanensis_seq1 100 I8199_R363_Anura_Pyxicephalidae_Cacosternum_platys_seq1 100 signal across I8191_R371_Anura_Pyxicephalidae_Cacosternum_albiventer_seq1 66 I10411_CAS_202097_Anura_Ranidae_Rana_Amietia_cf_tenuiplicata_seq1 100 I4419_CAS_236077_Anura_Microhylidae_Calluella_guttulata_seq1 I6438_AMCC_105559_Anura_Pyxicephalidae_Arthroleptella_bicolor_seq1 I8205_R569_Anura_Pyxicephalidae_Natalobatrachus_bonebergi_seq1

I7827_R410_Anura_Pyxicephalidae_Tomopterna_cryptotis_seq1

I7794_R527_Anura_Pyxicephalidae_Aubria_subsigillata_seq1

I6476_MCZ_A_140276_Anura_Bufonidae_Amietophrynus_camerunensis_seq1

I7801_R725_Anura_Pyxicephalidae_Pyxicephalus_adspersus_adspersus_seq1

I13340_PT440_Anura_Microhylidae_Callulops_personatus_seq1 I6439_MCZ_A_139541_Anura_Petropedetidae_Petropedetes_parkeri_seq1

I6428_AMCC_106956_Anura_Pyxicephalidae_Arthroleptides_martiensseni_seq1

I4390_MVZ_253198_Anura_Conrauidae_Conraua_crassipes_seq1 I6451_CAS_234799_Anura_Microhylidae_Glyphoglossus_molossus_seq1 I4416_CAS_218893_Anura_Phrynobatrachidae_Phrynobatrachus_leveleve_seq1 I6461_MVZ_226261_Anura_Pyxicephalidae_Cacosternum_boettgeri_seq1 I13351_PT507_Anura_Microhylidae_Metamagnusia_slateri_seq1 I6443_MCZ_A_136791_Anura_Phrynobatrachidae_Phrynodon_sandersoni_seq1 I7783_R1068_Anura_Ptychadenidae_Ptychadena_mascareniensis_seq1

I4418_CAS_219251_Anura_Ptychadenidae_Ptychadena_newtoni_seq1

I4415_CAS_230053_Anura_Odontobatrachidae_Odontobatrachus_natator_seq1

I7559_R846_Anura_Arthroleptidae_Arthroleptis_wahlbergi_seq1

I7557_R1020_Anura_Arthroleptidae_Arthroleptis_variablis_seq1

I13362_PT454_Anura_Microhylidae_Xenobatrachus_fuscigula_seq1I13338_PT439_Anura_Microhylidae_Barygenys_nana_seq1 the genome... I13346_PT452_Anura_Microhylidae_Genyophryne_thomsoni_seq1

I13355_PT455_Anura_Microhylidae_Oxydactyla_alpestris_seq1 I13343_PT428_Anura_Microhylidae_Copiula_oxyrhina_seq1

I13342_PT448_Anura_Microhylidae_Cophixalus_balbus_seq1

I13334_PT425_Anura_Microhylidae_Albericus_exclamitans_seq1

I10393_ABTC_50092_Anura_Microhylidae_Oreophryne_brachypus_seq1

I8225_R012_Anura_Hemisotidae_Hemisus_guineensis_seq1 I13341_PT441_Anura_Microhylidae_Choerophryne_proboscidea_seq1

I13348_PT424_Anura_Microhylidae_Hylophorbus_rainerguntheri_seq1 I4401_MVZ_249304_Anura_Hemisotidae_Hemisus_marmoratus_seq1

I4432_AMCC_105557_Anura_Brevicipitidae_Breviceps_macrops_seq1 I7707_R843_Anura_Hyperoliidae_Kassina_senegalensis_seq1

I4382_MCZ_138534_Anura_Brevicipitidae_Callulina_kisiwamsitu_seq1I7713_R838_Anura_Hyperoliidae_Semnodactylus_wealii_seq1

I6457_AMCC_124754_Anura_Hyperoliidae_Phlyctimantis_leonardi_seq1

I6474_CAS_168560_Anura_Brevicipitidae_Probreviceps_macrodactylus_seq1 I7704_R1129_Anura_Hyperoliidae_Heterixalus_luteostriatus_seq1 I7708_R1195_Anura_Hyperoliidae_Opisthothylax_immaculatus_seq1I7701_R1139_Anura_Hyperoliidae_Cryptothylax_greshoffi_seq1

I6475_MCZ_A_139760_Anura_Hyperoliidae_Afrixalus_fulvovittatus_seq1I4403_MCZ_136920_Anura_Hyperoliidae_Hyperolius_guttulatus_seq1

I7720_R306_Anura_Astylosternidae_Leptopelis_vermiculatus_seq1

I4375_CAS_168499_Anura_Arthroleptidae_Leptopelis_parkeri_seq1

I4428_AMCC_125880_Anura_Hyperoliidae_Alexteroon_obstetricans_seq1

I4376_AMCC_122837_Anura_Astylosternidae_Nyctibates_corrugatus_seq1

I6483_MCZ_A_139709_Anura_Astylosternidae_Scotobleps_gabonicus_seq1

I4442_AMCC_122836_Anura_Astylosternidae_Leptydactylodon_bicolor_seq1

I6437_MCZ_A_136806_Anura_Astylosternidae_Trichobatrachus_robustus_seq1

I6485_MCZ_A_137988_Anura_Arthroleptidae_Schoutedenella_sylvatica_seq1

I6477_MCZ_A_136805_Anura_Astylosternidae_Astylosternus_diadematus_seq1

I6478_MCZ_A_139626_Anura_Arthroleptidae_Cardioglossa_leucomystax_seq1

3.0 Inter-Ordinal Support Varies across Loci (RAxML “Best” Gene Trees)

Acauda n = 45

Batrachia n = 98 Procera n = 51 Support across 500 Bootstrap Replicates

Acauda Batrachia Procera Unbounded Support Using the AIC

100

∆ AIC ∆ 40

Acauda Batrachia Procera Unbounded Support Using the AIC

100

90 Constrain the basal

80 topology, then calculate AIC

70 for each model...

∆ AIC ∆ 40

Acauda Batrachia Procera Unbounded Support Using the AIC

100

90 Think of these as measuring

80 the strength of support

70 against alternative models.

∆ AIC ∆ 40

Acauda Batrachia Procera Backbone of the Amphibian Tree of Life

These topologies are largely concordant with previous studies...

With a few distinct exceptions... Backbone of the Amphibian Tree of Life

These topologies are largely concordant with previous studies...

With a few distinct exceptions... Conflict in the Frog Tree: Nasikabatrachus Conflict in the Frog Tree: Nasikabatrachus Conflict in the Frog Tree: Nasikabatrachus Different Methods Yield Different Topologies

ASTRAL Tree RAxML Tree *

78 100 100 100

100 100 A Framework for Testing Neobatrachian Relationships A Framework for Testing Neobatrachian Relationships Conclusions

• The illusion of “support” for topological hypotheses depends on how hard one looks.

• The bootstrap can help determine the direction of support, but may not be informative about its magnitude.

• Substantial discordance across loci exists at the base of the amphibian tree (and may not all be noise!).

• Genomic data and new statistical models are providing novel insights into evolutionary relationships of amphibians.

• More data ≠ easy answers (that are credible...). Why Blue Waters?

• Rigorously testing competing topological models across large numbers of genes is computationally demanding.

• Even embarrassingly parallel approaches (gene-by-gene AIC) overwhelm typical HPC clusters’ resources.

• MCMC sampling for rugged likelihood surfaces can be improved with large numbers of Metropolis coupled chains.

• For Bayes factor tests (with N taxa):

Markov chain Monte Carlo scales as N2

Hamiltonian Monte Carlo scales as N1.2 Acknowledgments Co-Authors/Collaborators Tissue Loans from Institutions: American Museum of Natural History (Darrel Frost, David Kizirian, Julie Feinstein) Alan R. Lemmon California Academy of Sciences (David Blackburn, Jens Vindum) Emily C. Moriarty Lemmon Florida Museum of Natural History (Pamela Soltis) University of Kansas Biodiversity Institute and Natural History Museum (Rafe Brown, Linda Trueb, Andrew Campbell) Elizabeth Scott-Prendini Louisiana State University Museum of Natural Science (Robb Brumfield, Donna Dittmann) Jeremy M. Brown Museum of Comparative Zoology (Jim Hanken, José Rosado, Breda Zimkus) Robert C. Thomson Museum of Vertebrate Zoology (Jim McGuire, Carol Spencer, Ted Papenfuss, Marvalee Wake, Sima Bouzid) Museum Victoria (Jane Melville, Joanna Sumner) Brice P. Noonan National Museum of Natural History (Kevin De Queiroz, Addison Wynn) R. Alex Pyron South African National Biodiversity Institute (Zoe Davids) Saint Louis Zoological Park (Jeffrey Ettling, Mark Wanner, Randall Junge) Pedro L. V. Peloso University of Michigan Museum of Natural History (Ronald A. Nussbaum, Gregory Schneider) Michelle Kortyna Yale Peabody Museum (Gregory Watkins-Colwell)

Justin D. Kratovil Tissue Loans from Individuals: J. Scott Keogh J.J. Apodaca, Alan Channing, Becky Chong, Guarino Colli, Tyler Frye, S. Blair Hedges, Elizabeth Jockusch, Christopher McNamara, Eric O'Neill, Todd Pierson, Steve Richards, Kelly Zamudio Stephen C. Donnellan

Rachel L. Mueller Fellowships: NSF Graduate Research Fellowship (3048109801), Blue Waters Graduate Research Fellowship (NSF/NCSA)

Christopher J. Raxworthy Funding: SSB Graduate Student Research Award, DEB-0949532 (DWW), DEB-1601586 (DWW, PMH) Krushnamegh Kunte

Santiago Ron

Sandeep Das

Nikhil Gaitonde

David M. Green

Jim Labisko

David W. Weisrock